1. Technical Field
The present invention relates to methods for determining the effects of a growth-altering agent on one or more microorganisms within a biological sample in general, and to methods for determining the effects of a growth-altering agent on individual microorganism colonies within a biological sample in particular.
2. Background Information
Effective patient treatment often requires an identification of microorganisms within a biological sample and a determination of the sensitivity of those microorganisms to growth-altering agents. Historically, biological samples have been taken and applied to or added to microbiologic growth media (called xe2x80x9cculturesxe2x80x9d), which were then examined and tested primarily on a macroscopic basis. In most conventional tests, a suitable growth medium is inoculated with a patient""s sample and subsequently incubated until there is visible evidence of microorganism growth. Most organisms require an incubation period of at least 18 to 24 hours to form visible colonies. The individual colonies start as a single, or a small cluster of microscopic cells or viable units (collectively referred to as colony-forming units or CFU""s) contained within the inoculum. After an initial lag period during which time the organism acclimates itself to its new environment and experiences little or no growth, the viable microorganisms settle into exponential growth: one cell will give rise to two cells in one generation, eight cells in three generations, sixty-four cells in six generation, and so forth until a visible colony is created.
If the inoculum contains a plurality of different microorganisms, each organism type will form its own characteristic colony, which may or may not be distinguishable from another. For most purposes, however, it is desirable to have only a single species of organism present within the organism growth. For example, if one wishes to test a biological sample for sensitivity to a particular antibiotic and the sample and subsequent culture contain multiple organism species, it may not be possible to determine the sensitivity of individual organism species within the culture to the antibiotic. To determine the sensitivity of individual organism species, it is necessary to make a xe2x80x9cpurexe2x80x9d culture (i.e., one that contains a single species of microorganism) by incubating the initial sample inoculum on a first solid growth medium and removing a single colony, or a group of identical colonies, from a first growth medium and plating it onto a second solid growth medium or forming a suspension if a liquid culture is used. A person of skill in the art will recognize the process is time consuming and generally requires a skilled technician.
In those instances where it is desirable to know the effectiveness of a growth-altering agent (e.g., antibiotics, growth promoting agents, nutrients, antiseptics, etc.) on an organism, prior art practice generally dictates the use of one of the following evaluative methods. In one method, a growth-altering agent is applied to a region of a solid inoculated growth medium prior to incubation and the organism growth in the applied region is evaluated or compared against organism growth in a region where growth-altering agent was not applied. A Kirby-Bauer plate test is an example of this type of macroscopic method. The Kirby-Bauer method includes incubating a growth medium until confluent growth forms over the growth medium. A region of growth medium bearing an effective growth-altering agent (antimicrobial) diffused out from a disk will not contain organism growth if the antimicrobial is effective in suppressing the organism. The size of the growth-free zone surrounding the disk is then compared to a reference to determine whether the organism is susceptible to the growth-altering agent in a clinically useful concentration. A second evaluative method involves adding a known amount of the growth-altering agent to a liquid medium that is inoculated with the organism to be tested. Turbidity testing is an example of this type of macroscopic method. A turbidity test measures the xe2x80x9ccloudinessxe2x80x9d of a liquid sample to determine the organism content of the sample. An increase in the turbidity of the sample indicates an increase in the organism content within the sample. A third evaluative method involves observing the effect organism growth has on a colored reagent that responds to one or more constituents or metabolic products of the growing organism. The information available from any of these macroscopic evaluation methods is, generally speaking, also macroscopic in nature; e.g., the growth-altering agent applied in a particular concentration either has or does not have an effect on the growth of the organism(s). Little or no additional information is available regarding, for example, the mechanism of death, whether the organism experienced septum formation, or any statistical information vis-a-vis the population of organisms within the culture.
One of the problems with the above macroscopic methods is test error that results from waiting until a visible layer or an acceptable concentration of organism develops. Organism colonies growing on or within a growth medium compete for food and as a result may be growth inhibited because of competition rather than because of a growth-altering agent. Those same organisms can also affect each other by their excretions and metabolic by-products. A more accurate analysis of the effect of a growth-altering agent on a particular microbe would be possible if such interference did not occur.
Another problem with macroscopic evaluation of an organism is the time required to produce meaningful results. As noted above, it is typically necessary to incubate an organism culture anywhere from 18 to 24 hours to produce a growth adequate for macroscopic evaluation (e.g., if the organism replicates every 20 to 60 minutes, there should be at least 20 generations of the organism). Practically speaking, however, generating a culture and analyzing it using conventional methods takes at least 48 hours because of handling, evaluation, etc. Because the rate of microbial growth is so rapid and the time for testing so great, patients suspected of having a microbial infection are often initially treated with a wide-spectrum antibiotic prior to the identification of the actual organism and its sensitivity. A more targeted treatment can be administered after the test data is received. A person of skill in the art will recognize, however, that wide spectrum antibiotics having the utility to provide more expeditious treatment are not favored over the more targeted treatments available with specific information. In fact, a wide spectrum antibiotic can be considerably more expensive and have more adverse side effects than a more targeted drug. There is also considerable concern today that the overuse of wide spectrum antibiotics might promote the development of antibiotic resistance within the organisms, consequently limiting their effectiveness.
In recognition of the problems associated with the time it takes to perform the above described macroscopic tests, a number of methods for rapidly determining antibiotic susceptibility have been proposed, including methods that examine individual organisms. These methods utilize the fact that susceptible bacteria may change their shape, size, or internal chemistry (or some combination thereof) when exposed to an antibiotic Some types of bacteria, however, do not detectably react to an antibiotic until after the propagation of the first few generations. Tests that only consider organisms in their first few generations, therefore, cannot provide useful information in every case and are considered to be ineffective unless the behavior of the organism is known in advance. An example of an analysis for a specific microorganism is proposed by Ledley (U.S. Pat. No. 5,922,282) for the determination of antibiotic susceptibility for mycobacterium tuberculosis (MTB). In the Ledley method, the DNA of individual organisms are altered by the addition of a plasmid which will cause the living organisms to fluoresce. The fluorescence of the organisms is compared before and after the addition of an antimicrobial agent to determine if the agent has extinguished the fluorescence and therefore killed the MTB organisms. This single-organism technique is similar to those previously published except for the means of creating fluorescence, and is only applicable to a narrow range of organisms. Another method for determining the effects of a growth-altering agent in a liquid broth is described in European Patent Specification No. EP 0 635 126 B1. In the European Patent Specification, image processing is used to determine changes in size, number, or shape of individual organisms to determine if there are effects from an antibiotic. A problem with this approach is that because it is performed in a liquid medium, we believe it to be impossible to analyze effects on a specific CFU or characteristics of that CFU""s such as the CFU""s replication rate.
Other methods for monitoring microbial growth and metabolism have been proposed that add agents designed to change color when exposed to microbial growth. Still other methods (as disclosed in U.S. Pat. Nos. 4,724,215; 4,720,463; and 4,856,073) examine microbial changes with a video camera. To the best of our knowledge, all of these methods are macroscopic in nature and consequently do not provide information about individual microbial colonies in their earliest stages and thus cannot provide reliable antibiotic resistance information in a very short period of time. They can provide only macroscopic information; i.e., whether or not the growth-altering agent had a detectable effect on the microbial growth.
It is also well known that all bacteria within-a given respond in the same manner to any particular growth-altering agent. There is a variation of resistance to growth-altering agents within any microbial population that is not easily quantified using any current technique. If this population data were routinely available, it may be possible to predict the likelihood of developing antibiotic resistance for the microbe in question and thus help determine the optimum length of treatment.
All previous methods of examining the effects of growth-altering agents on microorganisms of which we are aware can be grouped into two basic categories. The first group looks at the effects of organism growth visible to the naked, unaided eye; e.g., the formation of a visible colony or turbidity, or a color change associated with such growth. The second group relies on changes within single organisms within a liquid growth medium prior to logarithmic growth.
What is needed is a method for determining the reaction of a microbe to a growth altering agent, one that enables the microbe to be identified, one that can provide the aforementioned information in a fraction of the time it currently takes commercially available methods to provide it, and one that does not require visible macroscopic growth nor is limited to looking at single organisms.
It is, therefore, an object of the present invention is to provide a method for investigating the effects of growth-altering agents on microbial colonies within the first few hours of incubation.
It is another object of this invention to provide a method for identifying a microorganism by determining which nutrients or inhibitors affect its growth.
The present invention provides a method for determining the effects of a growth-altering agent on a microbial colony. The terms xe2x80x9cmicrobial colonyxe2x80x9d or xe2x80x9cmicrocolonyxe2x80x9d, as used herein refer to a microbial colony in its earliest stages of development, prior to its becoming readily visible to the naked eye. As used herein, the term xe2x80x9cgrowth-altering agentxe2x80x9d includes agents that will alter, inhibit, or enhance microbial growth. Examples of growth-altering agents include, but are not limited to antibiotics, antiseptics, nutrients, or growth promoting agents. Growth-altering agents can also be environmental type agents such as temperature, humidity, light, gaseous environment.
Each microbial colony analyzed under the present method is an individual microscopic colony that forms from a colony-forming unit (CFU) present within an inoculum. Meaningful information can be produced under the present method using colonies in a growth range that includes colonies that have doubled once from their CFU to those that have doubled twenty or so [fewer] times. The growth range of colonies capable of providing meaningful information under the present method may also be described in terms of growth time, colony size, or progeny. In most cases, the microbial colonies utilized under the present method generally cannot be seen by the naked human eye. Hence, the present method can be described as microscopic in contrast to prior art methods that macroscopically evaluate multiple microbial colonies contiguous with one another that are collectively large enough so as to be viewable by the naked eye. In addition, the present method reliably quantitates the growth characteristics.
The present method utilizes a solid or semi-solid growth medium and a growth-altering agent incorporated in at least a portion of the growth medium, and includes the following steps:
(a) inoculating the growth medium with an inoculum having one or more viable colony-forming units;
(b) incubating the colony-forming units in a manner likely to cause the colony-forming units to replicate into microbial colonies;
(c) quantifying one or more characteristics of one or more individual microbial colonies exposed to the growth-altering agent; and
(d) evaluating the quantified characteristics to determine the effects of the growth-altering agent on the individual microbial colonies.
During the quantifying step, characteristics of microbial colonies exposed to a growth-altering agent and in some instances characteristics of microbial colonies located in a control reference are quantified. A colony characteristic can be anything that can be quantified to provide meaningful data regarding the effects of the growth-altering agent on the colony. Colony characteristics typically useful for determining effects include, but are not limited to, colony area, perimeter, perimeter-to-area ratio, edge roughness, edge contour, and uniformity of colony density. The process used to quantify a characteristic can be varied to suit the determination at hand, the characteristic being quantified, and the level of specificity necessary to provide useful data. Quantifying processes include, but are not limited to, measurement, comparative, and inspection type processes. Quantifying is performed as a function of time. In all cases, the characteristics are quantified to ascertain change. In most cases, change is determined by comparing characteristics at two or more points in time (e.g., at T1, T2, T3, . . . , TN). In some cases, however, it may be possible to acquire meaningful information by quantifying a characteristic(s) at a single point in time. The number of times a microbial colony or colonies must be quantified will depend on the sufficiency of data collected; i.e., whatever number of times is necessary to make a clinically sufficient determination regarding the effects of the growth-altering agent. The mechanism used during the quantifying step is typically an imaging device (e.g., a digital camera, etc.) that produces an image with a clarity that is sufficient to allow the characteristics captured within the image to be measured, compared, inspected, or otherwise quantified. Other imaging devices such as bar scanners or flying-spot scanners may be used alternatively. Microcolony images can be utilized in real-time and/or saved.
The determination of the effects of the growth-altering agent on a microbial colony is made by evaluating the quantified characteristics. The manner in which the quantified characteristic is evaluated will, like the quantifying step, depend on determination at hand, the characteristic being quantified, and the level of specificity necessary to provide useful data. In some instances, the evaluation may only look at whether a quantified characteristic is present (e.g., whether colony growth exists, or whether the edge of a colony is rough, etc.) and that evaluation may take place using the characteristic data collected at a single point in time, or at a plurality of points in time. The determination of the effects the growth-altering agent has on the microbial colony in such cases, may be made based on the quantified characteristic alone. In other instances, the determination can be made by evaluating the quantified characteristic in view of a control reference.
A control reference may be any source of information that provides data useful in evaluating the characteristic of the microbial colony being considered. For example, if the inoculum contains a known organism, the evaluation could be performed using a control reference that provides data relating normal growth characteristics for that organism under similar environmental conditions; e.g., clinically developed data, etc. Alternatively, the evaluation can be performed using a control reference in the form of a section of growth medium inoculated with the same inoculum and incubated under similar conditions that is either not subjected to the growth-altering agent at all or is subjected to a different concentration of growth-altering agent. The characteristics of a colony in the control reference portion of the growth medium and the characteristics of a colony in the growth-altering agent applied region of the growth medium are quantified and evaluated in view of one another. In many instances, a comparative evaluation will yield data sufficient to make the determination. Other types of evaluation may be used alternatively.
The present method provides several significant advantages over the methods presently available for determining the sensitivity of an organism to a particular growth-altering agent. One distinct advantage is the speed by which a determination can be made regarding the effects of a growth-altering agent on an organism. This is particularly true when the present method is used to determine the effect that an antibiotic has on one or more organisms. As stated above, wide-spectrum antibiotics are often administered because of the initial lack of specific information from the patient""s sample. Wide-spectrum antibiotics are not favored over narrowly focused antibiotics because they can expose the patient to greater risk of adverse side effects, their overuse might promote the development of antibiotic resistance within the organisms, and they can also be very expensive. Using the present method, antibiotic sensitivity information can quite often be provided in two hours or less which is dramatically less than the typical turnaround possible using currently available methods. As a result, it is now often possible to effectively use narrowly focused antibiotics from the start, rather than initially treating the patient with a wide-spectrum antibiotic.
Several advantages stem from the fact that the present method determines the effects of a growth-altering agent on an organism using characteristic data collected from individual colonies. As stated above, the present method utilizes colonies very early in the incubation process (colonies that have doubled generally between two and twenty times) prior to any macroscopic aggregation of colonies within the culture that may not be of the same origin or the same type. One advantage that stems from individual colony data is that it is possible in some instances to use an xe2x80x9cimpurexe2x80x9d culture. Because data is collected from individual microscopic colonies there is a reduced need to separate different type colonies as is the case with macroscopic methods on solid media where it is likely that a variety of organisms would be aggregated into a macroscopic impure mass, or in a liquid suspension where it is difficult to distinguish the growth of one organism from another. Applying a growth-altering agent to a macroscopic impure mass would likely yield limited information because the effects of the growth-altering agent on the various different organisms would not be separable. Cultures made from urine or cerebrospinal samples are examples of possibly impure cultures where the present method may be used to evaluate different constituent organisms without first separating them.
Another advantage that stems from individual colony data is that it is possible to statistically analyze data pertaining to the effects of a growth-altering agent. Statistical data can be useful, for example, in determining an organism""s resistance to an antibiotic. Information pertaining to the organism""s antibiotic resistance can, in turn, provide valuable information regarding the optimum length of treatment. If, for example, an organism is found to be resistant to all but high concentrations of an antibiotic and the mechanism of susceptibility requires several generations to become effective, antibiotic treatment would be required for a longer period than in the case of a more sensitive organism which is immediately affected by the antibiotic.
Individual colony data also advantageously permits the identification of organism mutations that are uncharacteristically affected by the growth-altering substances. For example, the growth rates of the individual colonies can be statistically compared. If the range of growth rates exceeds the expected standard deviation of the control, it suggests that the organisms are more resistant than usual, and that resistance may be developing. It is important to emphasize that any microbial therapy must be directed to that of the most resistant organisms within the group, since these organisms will multiply even after the more sensitive organisms have been eliminated.
Another advantage of the present method is that it readily provides accurate growth-altering agent sensitivity information. A disadvantage of the Kirby-Bauer test is that there are a number of variables that affect the antibiotic concentration at any given point in the growth medium. Formulae have been published for calculating antibiotic concentrations based upon the clear zone size, but these formulae are rarely used and are considered to be approximations at best. One of the variables that can affect antibiotic concentration determination in a Kirby-Bauer is competition between adjacent organisms. In a standard plate test the organisms are in competition for food and therefore might experience inhibited growth as a result of the competition rather than as a result of the growth-altering agent. The organisms can also affect each other by their excretions and metabolic by-products. The present method avoids these types of interference by investigating the colonies shortly after inoculation, rather than waiting until the organisms have proliferated to the extent they can be seen by the naked eye.
Another advantage of the present method is its versatility. For example, the present method is capable of collecting data regarding the effect of a growth-altering agent on an organism from the moment the organism is applied to the growth medium. The present method is therefore not limited to looking at the first few generations of microbes, or only later generations that can be seen by the naked eye, but rather can be used for throughout the development of the organism. Another example of the versatility of the present method is that it can be used to investigate a particular concentration of growth-altering agent, or a gradient of concentrations of a growth-altering agent in a single test. The gradient approach avoids having to create multiple dilutions of an antibiotic, for example, to determine the minimum inhibitory concentration of that antibiotic for a particular organism. Still another example of the versatility of the present method is its ability to be used in the veterinary sciences.
The present method can also be used to facilitate the identification of particular organisms. For example, if the control reference in a particular test is a growth medium that does not contain a sugar, and the test area of the growth medium contains sugar, those organisms that can metabolize sugar will grow more rapidly in the test area than in the control area. One can therefore use a number of different growth-enhancing and growth-retarding substances to identify the organism in question.
Another advantage of the present method is its ability to provide information on effects, reactions, or lack of reactions that occur in statistically small numbers, but can nevertheless have significant implications vis-xc3xa1-vis the effectiveness of the growth-altering agent on the microorganism. The advantage stems from the fact that the present method evaluates microcolonies rather than macroscopic colonies. The number of microcolonies in a given area on a growth medium will be far greater than the number of macroscopic colonies in the same area, thereby providing a statistically more significant population for evaluation in that area. For example, it is well known that some bacteria have a low mutation rate, wherein anywhere from 1 in 104 to 1 in 106 could be resistant particular antibiotic under certain circumstances. If the growth of this resistant strain(s) is not detected, the strain will likely be falsely reported as susceptible to the antibiotic, when in fact the strain is resistant. The advantage provided by the present is perhaps best illustrated by example. If one were to use a video camera to image macroscopic colonies on a conventional agar plate (as is presently done with some instruments used to count colonies), there is not enough room on the agar plate to contain number of non-confluent macroscopic colonies statistically necessary to have an occurrence of the above described resistant bacteria. For example, a ten (10) centimeter diameter plate plated with macroscopic colonies spaced every two (2) millimeters can only hold about 2000 such colonies, which is clearly a statistically insufficient population for detection of such mutations. This statistically insufficient population is another reason why conventional agar plates used for antibiotic susceptibility are heavily plated so as to cause confluent growth. In contrast, under the present method the CFU""s that are incubated to become microcolonies can be seeded as close as ten microns (10xcexc) apart, which allows for the evaluation of a statistically adequate 104 microcolonies per square centimeter while allowing the evaluation to be performed prior to the microcolonies become confluent with one another.
Another advantage of the present method is that it can also be used to detect the presence of growth-altering agents. For example, there is utility in determining the presence of antibiotics or other growth-altering agents within a sample of milk. Current milk testing procedures require that the type of antibiotic being tested for be known up-front; i.e., the tests are tailored to particular antibiotics. Under the present method, milk is incorporated into a growth medium and an inoculum is inoculated into the growth medium. The inoculum is selected as one that is likely to be affected by the presence of a growth-altering agent (e.g., antibiotic residue) within the milk. If the growth of microcolonies originating from the inoculum is different from normal growth under the conditions at hand, then the presence of antibiotics known or unknown is likely within the sample of milk.
These and other objects, features and advantages of the present invention will become apparent in light of the detailed description of the best mode embodiment thereof, as illustrated in the accompanying drawings.